PDGF was first recognized as a component of platelet .alpha. granules, which had growth promoting activity for smooth muscle cells and fibroblasts (Heldin and Westermark, Cell Regul 1: 555-566 (June 1990)). It has also been implicated in the stimulation of connective tissue--derived cells in vitro (Ostman et al., J. Biol. Chem. 263(31): 16202-16208 (November 1988)), as the major mitogenic protein for mesenchymal cells (Murray et al., U.S. Pat. Nos. 4,889,919 and 4,845,075), and as an inducer of cell multiplication and DNA synthesis in cultured muscle cells, fibroblasts and glial cells (Kelly et al, PCT Application WO90/14425 (Nov. 29, 1990)). It has also been shown to be involved in the wound healing response (Ross et al., N. Eng. J. Med. 295:369 (1976)), and may be involved in a causative role for the development of proliferative lesions of atherosclerosis (Ross) supra. Others have suggested that this molecule may be a mediator of tumor development as well as in nonmalignant proliferative disorders (Heldin et al., supra).
The PDGF molecule has been very well characterized. It is known to exist as a heterodimer of an "A" chain and a "B" chain, connected to each other via disulphide bonds. The dimer, sometimes referred to as "PDGF-AB", has a molecular mass of about 30 KDa. Amino acid sequences are known for both the A and B chains, as shown, e.g., by Murray et al., U.S. Pat. Nos. 4,889,919 and 4,845,075, the disclosures of which are incorporated by reference. The mature chains contain slightly more than 100 amino acids, and are about 60% homologous. Heldin et al., supra.
Dimers PDGF-AA and PDGF-BB have been produced via recombinant means, and have also been isolated from natural sources (see Murray et al., Supra; Heldin et al., Supra). The various dimers, or "isoforms" differ in functional properties and secretory behavior.
The mechanism by which PDGF acts on cells has received intensive scrutiny, and it has been established that there are two receptors for PDGF, the ".alpha." and ".beta." receptors. The .alpha. receptor binds all isoforms, whereas the .beta. receptor does not bind PDGF-AA, binds PDGF-AB with low affinity, and PDGF-BB with high affinity (Heldin et al., supra; O stman et al., supra). The .alpha. receptor is synthesized as a 140 KDa precursor protein which matures to one of 170 KDa, and the .beta. receptor is recognized as a precursor of 160 KDa, and a mature molecule of 180 KDa. cDNA for both receptors have also been isolated (Heldin et al., supra; Kelly et al., supra).
The receptors both comprise five immunoglobulin like domains (extracellular portion), and intracellular portions containing protein tyrosine kinase domains with characteristic insert sequences which have no homology to kinase domains (Yarden et al., Nature 323: 226-232 (1986); Matsui et al., Science 243: 800-803 (1989)); Claesson-Welsh et al., PNAS 86: 4917-4921 (1989). When PDGF binds to these receptors, dimerization of the receptor molecules is induced, followed by kinase activation and autophosphorylation of the receptors (Heldin et al., J. Biol. Chem. 264: 8905-8912 (1989); Seifert et al., J. Biol. Chem. 264: 8771-8778 (1989); Bishayee et al., J. Biol. Chem. 264: 11699-11705 (1989)).
The diverse actions of PDGF and its suggested involvement in disease states would indicate that the use of agonists and antagonists may be useful in defining the action of PDGF and of alleviating some of the disorders. These molecules, using the definitions employed by Kelly et al., supra, either mimic the effect of PDGF (agonists), or block the interaction of receptor and ligand (antagonists).
The art has long recognized that agonists and antagonists for various materials exist, and Kelly et al., via their discussion, de facto assume that these exist for PDGF. Review of the literature indicates, however, that no proteinaceous agonists and antagonists to PDGF are taught. For the reasons described supra, it would be desirable to have such material available.
The two patents to Murray et al., cited supra discuss potential amino acid substitution of cysteine residues in the monomeric chains, provided that these substitutions do not destroy the biological activity of the molecules. The '919 patent generally teaches modifications of PDGF AA molecules. Neither reference teaches that modified dimers of PDGF have antagonistic activity against wild type PDGF.
It has now been found that substitutions within the amino acid chain of PDGF monomers leads to the production of antagonists to PDGF-BB. As PDGF-BB is implicated in the transformation of cells, the antagonists have value in a therapeutic context, as well as in various other milieux, as described in the disclosure which follows.